High-throughput instant quantification of protein expression and purity based on photoactive yellow protein turn off/on label

Abstract

Quantifying the concentration and purity of a target protein is essential for high-throughput protein expression test and rapid screening of highly soluble proteins. However, conventional methods such as PAGE and dot blot assay generally involve multiple time-consuming tasks requiring hours or do not allow instant quantification. Here, we demonstrate a new method based on the Photoactive yellow protein turn Off/On Label (POOL) system that can instantly quantify the concentration and purity of a target protein. The main idea of POOL is to use Photoactive Yellow Protein (PYP), or its miniaturized version, as a fusion partner of the target protein. The characteristic blue light absorption and the consequent yellow color of PYP is absent when initially expressed without its chromophore, but can be turned on by binding its chromophore, p-coumaric acid. The appearance of yellow color upon adding a precursor of chromophore to the co-expressed PYP can be used to check the expression amount of the target protein via visual inspection within a few seconds as well as to quantify its concentration and purity with the aid of a spectrometer within a few minutes. The concentrations measured by the POOL method, which usually takes a few minutes, show excellent agreement with those by the BCA Kit, which usually takes ∼1 h. We demonstrate the applicability of POOL in E. coli, insect, and mammalian cells, and for high-throughput protein expression screening.

Keywords: color tag; high-throughput protein expression screening; protein expression test; protein purification; protein purity estimation; protein quantification.

Figure 1

Schematic representation of the POOL system and comparison with the conventional PAGE method. (A) POOL without chromophore is co-expressed with target protein. Upon addition of a precursor of chromophore, POOL exhibits yellow color, thereby signaling the expression of the target protein. (B) POOL reduces time-consuming steps that take over several hours when using PAGE.

Figure 2

Quantification of the target protein with/without the POOL system by observing the color change and UV–Visible spectroscopy. This figure shows a comparison of the color difference and UV–Visible spectra before and after adding a precursor of chromophore. (A) The color change with/without POOL. The first corresponds to SUMO–POOL fusion protein before adding a precursor of chromophore. The second corresponds to SUMO–POOL fusion protein after adding a precursor of chromophore. The third corresponds to a target protein not fused with POOL after the addition of a precursor of chromophore. (B) UV–Visible spectra corresponding to (A). The blue, red and black curves correspond to the first, second and third cases of (A), respectively. (C) Standard reference solutions used for estimating the concentration. (D) Comparison of the protein concentration measured by the BCA method and POOL method. The slope is 0.99 with a mean error of ∼4%.

Figure 3

Eluted fractions (middle) of SUMO–POOL fusion protein via ion-exchange chromatography, their corresponding PAGE gels (upper) and UV–Visible spectra (bottom), and the merits and disadvantages of PAGE and POOL.

Figure 4

Optimization of the expression condition by high-throughput protein expression screening aided by POOL for human calmodulin. To determine the best expression condition of human calmodulin and to study the efficiency of POOL, the E. coli lysate with the constructs with and without POOL were tested. The color change is not observed in the wells containing cell lysate without POOL. The optimal expression condition can be identified simply by the addition of a precursor of chromophore. The measured concentration is shown for each well.

Figure 5

Results of calorimetric screening of PYP deletion mutants. The color change of 12 deletion mutants upon adding a precursor of chromophore is shown. Persistent color change was observed for N5, N10, and N18, but the rest of the N-terminal deletion mutants and all C-terminal deletion mutants showed weak color change that gradually faded. The N27C18 deletion mutant can be still used to check expression of the target protein as it shows color change for a few minutes.